The objectives of this project are 1) to characterize both the cellular and bacterial components of mycobacterial entry into and survival within host cells and 2) to characterize the state of "dormant" Mycobacteria and identify bacterial determinants necessary for long-time survival in the host. We have begun our investigations by exploring the usefulness of M. marinum and M. ulcerans, as a model for M. tuberculosis. These mycobacterial species are taxonomically a part of the M. tuberculosis complex. Initial work has focussed on understanding the relationship between M. marinum and host cells. We have found that M. marinum have the ability to adhere to, enter and replicate within macrophage, epithelial and fibroblast cell lines. Electron micrographs demonstrate that early in infection intracellular organisms are found within membrane bound compartments. Long-term infection of fibroblast and macrophage cell lines results in detachment of cells from the monolayer. In contrast to M. marinum, M. ulcerans neither replicates nor adheres to host cells. However, both live bacteria as well as sterile filtrates from M. ulcerans cultures are cytotoxic for host cells. We have found that cytotoxicity is a characteristic of stationary phase cultures only. Further investigation will be needed to define the intracellular compartment within which mycobacteria replicate. We plan to identify genetic determinants responsible for cell entry, intracellular growth and cytotoxicity. With the completion of a P-3 facility required for working with virulent Mycobacterial tuberculosis, we will extend this work by looking at the interaction of M. tuberculosis with host cells. All M. marinum examined so far contain a 3 kb and 5 kb plasmid. These are the only plasmids identified from bacteria in the M. tuberculosis complex. We plan to explore the use of these plasmids for developing a genetic system for use in M. tuberculosis.